Mitochondria are dynamic multifunctional organelles that play pivotal biochemical roles in cell's homeostasis and survival. In addition to generating ATP, mitochondria are also important for Ca2+ signaling, neurotransmitter metabolism, and reactive oxygen species (ROS) signaling. Mitochondrial dysfunctions represent a point of convergence for almost all major neurodegenerative disorders, as well as cases of bioenergetics failures, such as ischemia-reperfusion injury, trauma, and toxic exposures. Yet, the exact mechanisms via which dysfunctional mitochondria contribute to the pathology of these conditions remains unknown. In order to maintain homeostasis and minimize the effect of damaged proteins and organelles, the cellular proteome is constantly degraded to make way for the new versions. Post-mitotic long-lived cells, such as neurons, are particularly sensitive to these processes since they cannot dilute the macromolecular damage though cell division. However, in our previous research we identified intracellular proteins in the brain with exceptionally long-life spans that we termed extremely long-lived proteins (ELLPs). Due to their persistence, ELLPs are likely to accumulate damage over longer periods and thus create a previously discounted layer of cell's vulnerability. Intriguingly, we found that a small subset of brain's mitochondrial proteome is also long-lived and persists for the entire lifetime of a mouse. We hypothesize that these newly identified mitochondrial ELLPs (mito-ELLPs) play an underappreciated role in the processes of neurodegeneration and cell's response to stress and injury. The goal of this research proposal is to identify and characterize mitochondrial ELLPs in mammalian neurons. Using metabolic stable isotope pulse-chase labelling, together with high-resolution shotgun mass spectrometry (MS)-based proteomic analysis, and custom bioinformatics strategies, we will define the mitochondrial long-lived proteome in mouse brain extracts and primary neuron cultures. Furthermore, we will determine whether mito-ELLPs localize preferentially to damaged, unfit, or aged mitochondria by combining advanced live cell fluorescent techniques and organelle sorting. We will further delineate subcellular localization of mito-ELLPs by co-localization studies using markers of synapses, ER, MAMs, and mtDNA translation. Lastly, we will investigate whether pharmacological treatments known to affect mitochondrial function affect the long- lived proteome. Insights from these experiments will significantly advance our understanding of the role of mito- ELLPs in neurons and could lead to new targets for potential therapeutic interventions for a myriad of neurological disorders.